Cytotoxic clerodane diterpenoids from the roots of Casearia barteri Mast.

A study of diterpenoids as active ingredients against cancer from the active roots extract of Casearia barteri Mast. (IC50 = 1.57 μg mL−1) led to the isolation of six new clerodane diterpenoids, named as barterins A–F (1–6) alongside seven known compounds, caseamembrin A, caseamembrin E, casearlucin A, graveospene G, N-trans-feruloyltyramine, N-cis-feruloytyramine and sitosterol-3-O-β-D-(6-O-palmitoyl)-glucopyranoside. Their structures were elucidated based on NMR spectroscopic data and mass spectrometry. The absolute configurations of 1–6 were established by the time-dependent density functional theory (TDDFT), electronic circular dichroism (ECD) calculations and experimental data analysis. The cytotoxic effects of compounds 1–6 were evaluated against a human cervix carcinoma cell line KB-3-1. Barterins A–D (1–4) showed cytotoxic effects against the KB-3-1 cell line with IC50 values ranging from 1.34–4.73 μM.


Introduction
Natural products have been a cornerstone of drug discovery given their considerable chemical diversity, which has proven invaluable in the discovery of new anti-cancer agents. 1 As the global cancer incidence continues to rise, the search for effective treatments remains a critical challenge.3][4] Belonging to the family Flacourtiaceae, the genus Casearia includes a group of about 180 plant species widely spread in tropical and subtropical areas of Africa, Asia, Australia, North and South America and the Pacic islands. 5They are abundantly mentioned in traditional medicine for the treatment of diarrhea, skin lesions, ulceration, tropical leprosy, herpes, snake bite and fever. 6Numerous investigations on species of the genus Casearia Jacq.][9][10][11] Moreover, biological assays have revealed that these diterpenes possess cytotoxic, antibacterial, antifungal and DNA-modifying properties. 5asearia barteri Mast. is a small to medium-size tree, up to 20(−40) m high, present in tropical forest of Cameroon and Nigeria.The twigs and stem barks are chewed for sore gum and teeth cleaning. 12This high chemical and pharmacological potential as well as the contribution of the Casearia genus to traditional medicine motivated our choice of the study presented here.

Results and discussion
The crude extract derived from maceration of C. barteri roots in methanol led to further semi-pure extracts aer fractionation with petrol ether-acetone solvent system of increasing polarity.These were puried using chromatographic columns with various stationary phases, leading to the isolation of thirteen compounds, including six new derivatives, barterins A-F (1-6) (Fig. 1) along with seven known compounds identied as caseamembrin A (7), caseamembrin E (8), 13 the mixture of casearlucin A (9) 14 and graveospene G (10), 11 N-trans-feruloyltyramine (11), N-cis-feruloytyramine (12), 15 (−)-b-sitosterol-3-O-b-D-(6-O-palmitoyl)glucopyranoside (13) 16 using their spectroscopic data by comparing with those reported in the literature.1).The decoupled broadband 13 C NMR spectrum of 1 exhibited resonances of 31 carbon atoms including those of 2-methylbutanoyloxy, butanoyloxy, and acetyloxy groups established from the observation of the following carbon signals (d C 175.8, 41.9, 27.7, 17.1, 12.1; 173.1, 36.7,18.9, 13.9; 169.6, 21.9) (Table 2).The remaining 20 carbons have been assigned to those of the diterpene-type skeleton with the help of DEPT and HMQC spectra having three double bonds including one terminal olen (d C 121.7, 147.5, 136.0, 130.9, 111.0; 142.5) as well two acetal carbons (d C 96.4, 98.2).8][19] 2D homo-and hetero-nuclear correlations were further used to conrm the skeletal type of compound 1 and identify the position of its functional groups and substituents (Fig. 2).A tri-substituted double bound was located at position C-3/C-4 of the decalin ring system of diterpene from long range hetero-nuclear correlation of the proton signal at d ).Furthermore, the butanoyl substituent was inferred to be attached at position C-18 from the 3 J correlation between the proton signal at d H 6.69 (H-18)  and the carbonyl at d C 173.1 (C-1 00 ).In the same way, the correlation between the proton signal at d H 6.47 (H-19) and the carbonyl at d C 169.6 (C-1 000 ) was used to attach the acetoxyl group at position C-19.Following the same rationale, the 2-methylbutanoyloxy was attached at position C-2 considering the deshielding effect of the proton H-2 (d H 5.40) attributable to the attractive mesomeric effect of the ester function, in agreement with data reported for other diterpenoids from the genus Casearia in the literature. 2,10An additional hydroxy group was located at C-6 using correlation between H-19 (d H 6.47) and C-6 (d C 72.9), and from H-10 (d H 2.44) to C-6 (d C 72.9).The E-conguration of the double bond C-12/C-13 as well as the transoid geometry of the conjugated double bond were established from NOESY interactions between H-11a,b/H-16/H-15 and H-8/H-12/H-14 and from the 13 C NMR chemical shi of the allylic carbon C-11 at d C 31. 1. 11 Similarly, the relative conguration of the decalin ring system was discussed aer examination of spatial correlations between H-17/H-10 and H-2/ H-3/H-18/H-19/H-6/H-20/H-8.From these evidence, the decalin ring was established as cis fused with proton H-10 and methyl H-17 both a-oriented while protons H-2, H-6, H-18, H-19 and methyl H-20 adopted a b position (Fig. 3).The relative conguration (2R,5S,6S,8R,9R,10S,18R,19S) obtained from the analysis of spatial correlations was used as a model for the ECD calculations.The similarities between the negative Cotton effect at 215 nm of the experimental and theoretical ECD curves (Fig. 4) allow us to assign the absolute conguration 2R,5S,6S,8R,9R,10S,18R,19S for compound 1.These observations were in agreement with the stereochemistry of graveospene A. 11 Compound 2 was puried as a colorless oil.The molecular formula of compound 2 was deduced to be C 30 H 44 O 9 from the sodium adduct ion [M + Na] + observed on its HRESIMS spectrum at m/z 571.2878 (calcd for C 30 H 44 NaO 9 , 571.2877) and its NMR data.The exhaustive analysis of the 1D NMR spectra revealed that compound 2 is analogous to compound 1, notably due to the presence of the same substituents The position of the aldehyde function at C-14 was further conclusive aer interpretation of the HMBC correlations of its proton (d H 9.40, H-14) with olenic and methyl carbons respectively at d C 153.5 (C-12), 142.0 (C-13) and 9.5 (C-16) as well as those between methyl H-16 with carbon C-12, C-13, C-14 (Fig. 2).The double bond C-12/C-13 was identied as E-   The same relative conguration for compound 4 as that of 1 was corroborated from H-20/H-8/H-11a/H-16/H-15a,b/MeO-14, H-2/ H-3/H-18/H-19/H-6/H-1a.Similarly, the orientation of the proton H-14 in b position was deduced from NOESY cross peaks from H-12/H-20/H-14.The experimental ECD curve was consistent with the calculated ECD (Fig. 4) for the conguration 2R,5S,6S,8R,9R,10S,14R,18R,19S establishing thus the absolute conguration of compound 4.
Compound 5, obtained as a brownish oil with a molecular formula C 31 H 46 O 9 based on the presence of a HRESIMS ion peak at m/z 585.3035 [M + Na] + (calcd for C 31 H 46 NaO 9 , 585.3034).Compound 5 contains nine double bond equivalents and is like compound 1 described above with respect to the number of carbon atoms, but with an additional oxygen atom.In addition to the signals related to the identical acyl substituents present, a comparative analysis established that the basic skeleton of compound 5 belongs to isozuelanin, with two terminal double bonds at the level of the lateral chain of clerodane-type diterpenes.Indeed, the DEPT 135 of compound 5 spectrum exhibited resonances of two olenic methylene carbons at d C 114.7 and 115.2.The typical b-monosubstituted diene of the isozuelanin skeleton was conrmed by interpretation of the HMBC correlations of both terminal protons at d H 5.11 (H-16) and 5.45 (H-15) with olenic carbons at at d C 151.9 (C-13) and 138.2 (C-14).Beyond this aspect of position isomerism, compound 5 was found to have an additional oxygen atom as outlined above, which resulted in the presence of an   The absolute conguration 2S,5S,6S,8R,9R,10S,18R,19S was inferred from the comparison between theoretical and experimental ECD curves, with a negative Cotton effect at 228 nm (Fig. 4).

Cytotoxic activity
Cervix cancer is the fourth most diagnosed cancer in women worldwide, with more than half a million new cases and 311 365 deaths in 2018, with nine-tenths of these being among women living in low-and middle-income countries. 20A large number of plants and plant-derived principles has demonstrated anticancer activity through selective cytotoxicity towards tumour cells.To bring our contribution to the search for new anticancer agents, we evaluated the cytotoxic potential of diterpenes 1-6 and the crude extract towards the cervix carcinoma cell line KB-3-1 (a HeLa subclone) with griseofulvin as standard and using the MTT assay as previously described. 21The crude extract exhibited strong cytotoxic activity with IC 50 of 1.58 mg mL −1 , in agreement with the range dened by the National Cancer Institute in US, which considers an extract to be active when the IC 50 value aer incubation between 48 and 72 h, is less than 20 mg mL −1 . 22This corroborates the results obtained on   3).It is noteworthy that these full one of the criteria attributed to potential plant-derived anti-cancer candidates with an IC 50 value less than 10 mM. 25 The high activity of compounds 2 and 6 could be related to the aldehyde function at position 14.However, further analysis needs to be carried out to better elucidate the inuence of this chemical function, as no bioassay results or computational analysis on the cytotoxic activity of analogue compounds is available in the literature.

Experimental section
General experimental procedures UV spectra were obtained using a Hitachi UV 3200 spectrophotometer, and IR spectra with a using JASCO 302-A spectrometer (Thermo Scientic, Waltham, MA, USA).ECD spectra were obtained on a JASCO J-715CD spectrometer (JASCO Corporation, Tokyo, Japan).Optical rotations were measured on a JASCO DIP-3600 digital polarimeter (JASCO, Tokyo, Japan) at 23 °C. 1 H, 13 C and 2D NMR spectra were recorded at room temperature using a Bruker DRX-600 spectrometer (125 MHz for 13 C and 500 MHz for 1 H, Bruker, Germany) to provide chemical shis that are expressed in ppm relative to tetramethylsilane (TMS).ESI-Mass spectra were obtained with an Agilent 6220 TOF LCMS mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).Column chromatography was performed using silica gel of 70-230 mesh (Merck, Darmstadt, Germany) and aluminum plates precoated with silica gel 60 F254 (Merck, Darmstadt, Germany) were used for thin layer chromatography.TLC spots were visualized under UV light at 254 or 365 nm, followed by spraying with 20% aqueous H 2 SO 4 spray and heating.Normal phase medium pressure liquid chromatography (MPLC) was run using Reveleris X2 Flash Chromatography System equipped with an UV-vis and ELSD detectors (Buchi, Switzerland).

Plant material
The roots of Casearia barteri Mast.were collected in March 2016 from Bangangté, West Province, Cameroon.The botanical identication was made by the botanist Mr Nana Victor at the National Herbarium of Cameroon, where a voucher specimen (no.65823 HNC) were already available.

ECD calculations
The calculated ECD spectra of new compounds were performed as previously reported. 26,27Systematic conformational searches were performed rstly using MOE soware and conformers under 3.0 kcal mol −1 were optimized using the DFT method at the B3LYP/6-31+G(d,p) level (Gaussian 09). 28A second optimization was performed using the DFT method at the B3LYP/6-311+G(d,p) level, and a polarizable continuum model (IEFPCM, solvent: acetonitrile) was applied to mimic the effects of the solvent used in the experimental ECD spectra.Theoretical ECD spectra were simulated for those conformers falling above 1% population threshold aer applying Boltzmann distribution.Time Dependant Density Functional Theory (TDDFT) was used at the CAM-B3LYP/6-31+G(d) level of theory (IEFPCM, solvent: acetonitrile).The ECD curves were extracted using SpecDis 1.61 soware. 29The overall ECD curves of all the compounds were weighted by Boltzmann distribution aer UV correction.

Conclusions
Six new clerodane-type diterpenoids, barterins 1-6 as well as four knowns were described from the roots extract of Casearia barteri.These isolates were found to share the same skeleton as the previous ones reported from the genus Casearia, enhancing their character as chemotaxonomic markers of this genus.Besides, their structural diversity, the reported diterpenoids are also endowed with signicant cytotoxic activity, such as barterin B and E which presented the pronounced inhibition activity (IC 50 = 1.34, 1.54 mM respectively) while the crude extract exhibited also a strong activity (IC 50 = 1.58 mg mL −1 ).These observations suggest that Casearia barteri should be given considerable attention, especially to better understand the mode of action in the perspective of the search for alternative treatment for cervical cancer.
(d H 3.54/d C 90.5) together with the loss of the signals from the terminal double bond C-14/C-15.Strong 1 H-1 H 3 J COSY correlations were observed between the oxymethine and the oxymethylene protons and their position at C-14 and C-15 were inferred from HMBC cross-peaks from the methyl protons at d H 1.54 (H-16) to carbons C-12 (d C 128.1), C-13 (d C 135.6) and C-14 (d C 90.5) and between the oxymethine proton at d H 3.58 (H-14) and carbon C-12, C-15.Furthermore, correlations of the oxymethine proton H-14 with the methoxy carbon at d C 56.6 evidenced the position of the methoxy at C-14.

Table 1 1
H NMR data for compounds 1-6 (600 MHz, d in ppm, J in Hz) Compound 4 was also isolated as a brownish oil.Its HRE-SIMS in positive mode having the sodium adduct peak at 617.3292 [M + Na] + (calcd for 617.3296 for C 32 H 50 O 10 Na), suggested a molecular formula of C 32 H 50 O 10 , corresponding to eight degrees of unsaturation.NMR data comparison revealed that compound 4 was similar to 1, except for the presence of a methoxy (d H 3.20/d C 56.6), an oxymethylene (d H 3.58, 3.45/d C 64.8) and an oxymethine group experimental one (Fig.4) and this unambiguously led to the assignment of the absolute conguration for compound 3.
24However, few studies have been done towards cervix cancer cell lines, such as the one performed by Silva, which indicated a moderate activity of the essential oil of C. sylvestris towards HeLa cells with IC 50 = 63.3 mg mL −1 .24Compounds 1, 2, 3 and 6 also had pronounced activity with IC 50 values of 2.52 mM, 1.34 mM, 4.73 mM, and 1.54 mM, respectively, compared to the control griseofulvin (IC 50 = 19.3mM) while compounds 4 and 5 were inactive (IC 50 > 200 mM) (Table